The present invention relates to methods and compositions for inhibiting the infectious activity of viruses by the use of lectins. More specifically, it relates to the use of lectins obtained from Sambucus nigra for inhibiting the activity of enveloped viruses.
Viruses may be classified in a number of different ways. However, one major division is between those viruses wherein the virion comprises an envelope surrounding the nucleocapsid and those which do not. Among the viruses wherein the nucleocapsid is enveloped are: (1) DNA viruses such as Herpesviridae including herpes simplex, cytomegalovirus, varicella zoster, Epstein Barr and other viruses affecting horses, chickens and cows; Poxviridiae including smallpox, vaccinia, human monkeypox, sheep, bird, swine pox viruses and Iridoviridae; (2) RNA viruses such as Togaviridae including alphaviruses such as Sindbis, equine and encephalitis viruses: flaviviruses such as Yellow fever and dengue viruses; rubiviruses such as rubella and pestiviruses; Arenaviridae, Orthomyxoviridae, including influenza types A, B and C viruses; Paramyxoviridae such as parainfluenza (types 1, 2, 3 and 4); respiratory syncitial, mumps, measles and Newcastle disease viruses; Coronaviridae such as coronavirus; Rhabdoviridae; Retroviridae such as oncovirinae, spumavirinae and lentivirinae; Bunyaviridae such as bunyavirus; and certain unclassified viruses such as hepatitis A, hepatitis B, non A non B heptatis viruses, as well as certain viruses connected with tumor production and disorders such as acquired immune deficiency syndrome.
Enveloped animal viruses are characterized by the presence of a membrane consisting of a lipid bilayer with glycoprotein projections, or spikes, on the outer surface of the viral envelope. These spikes consist of virus-coded glycoprotein molecules which are essential for viral infectivity and replication. The antigenic specificity of the virus is also determined by these molecules.
The viral glycoproteins are involved in the early interactions between the virion and the cell, i.e. adsorption to the cell surface and penetration of the virion into the cell. The exact sequence of events in the penetration of enveloped viruses into cells is still not clear. However, in the case of the paramyxo- and myxoviruses, it is clear that fusion of viral and cell membranes is involved in the penetration step and that penetration is mediated by a viral glycoprotein. Specific cleavage of the viral glycoprotein activates the ability of the virus to initiate infection.
Lectins are a group of proteins capable of binding specifically to carbohydrate moieties on various cell surfaces. The interaction of a few lectins with glycoproteins on virus envelope has been investigated. It has been found that Concanavalin A and Ricinus communis agglutinin, especially the former, do interfere with the activity of a number of viruses. The toxicity of Concanavalin A and Ricinus communis agglutinin, however, precludes their clinical use (the latter is one of the most toxic substances known). Other less toxic lectins have been investigated with mixed results.
The ability of Concanavalin A to interfere with viruses has been described in many articles in the literature. For example, Calafat and Hageman in J. Gen. Virol 14: 103-106 (1972) describe the binding of murine RNA tumor viruses with Concanavalin A. Birdwell and Strauss in J. Virol. 11: 502-507 (1973) reported that Concanavalin A and Ricinus communis agglutinin agglutinate Sindbis virus. Stewart et al. in Proc. Nat. Acad. Sci. USA 70: 1308-1312 (1973) describe the use of Concanavalin A to selectively agglutinate murine and avian oncornavirions, especially Friend virus. Okado and Kim in Virol. 50: 507-515 (1972) showed that the activity of the enveloped viruses of Sendai virus and herpes simplex virus was destroyed by Concanavalin A, but that this lectin had no effect on unenveloped polio virus.
Ito and Barron in J. Virol. 13: 1312-1318 (1974) report that whereas Concanavalin A inhibited the infectivity of herpes simplex virus type 1, phytohaemaglutinin-P. wheat germ agglutinin and pokeweed mitogen had no such effect. They also reported that Concanavalin A inactivated herpes simplex virus type 2, pseudorabies virus and vesicular stomatitis virus, but that there was no such effect on vaccinia, simian adenovirus SV 15 and echovirus type 6. The data suggested that Concanavalin A blocked the binding sites on the virion envelope.
Ito and Barron in J. Gen. Virol. 33: 259-266 (1976) showed that while phytohaemagglutinin-P failed to inactivate herpes simplex virus, it did enhance the inactivation effected by Concanavalin A.
Finkelstein and McWilliams in Virol. 69 570-586 (1976) reported the effects of a variety of lectins on various viruses. In particular, they reported that phytohaemagglutinin-P, Concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin were effective in inhibiting the growth of Sindbis virus and vaccinia virus in chick embryo and Vero cells. They also reported that soybean agglutinin and pokeweed mitogen had little effect on the viruses.
Stitz et al in J. Gen. Virol. 34: 523-530 (1977) reported studies of the effect of Concanavalin A on the final stages of replication of fowl plague virus and Newcastle disease virus.
They suggested that a lattice was formed comprising virus particles and Concanavalin A molecules which prevented release of virions.
Cartwright in J. Gen. Virol. 34: 249-256 (1977) concluded that the effect of Concanavalin A in preventing the replication of mature vesicular stomatitis virus was due to blockage of the glycoprotein receptor sites on the cell by the lectin.
Ito et al in Arch. Virol. 57: 97-105 (1978) report that human cytomegalovirus (a herpesvirus) can be inactivated by phytohaemagglutinin whereas herpes simplex virus is not and that wheat germ agglutinin and pokeweed mitrogen were ineffective against both viruses.
Urade et al in the Arch. virol. 56: 359-363 (1978) reported that the infectivity of wild type rubella viruses was destroyed by Concanavalin A whereas this lectin had little effect on rubella pi variants. The authors concluded that the difference resulted from a difference in carbohydrate content in the structure of the envelopes.
Delagneau et al reported in Ann. Virol. (Inst. Pasteur) 132 E.: 461-471 (1981) that certain lectins, phytohaemagglutinin-Els, wheat germ agglutinin, Ricinus communis agglutinins, Lens culinaris agglutinin and to a lesser extent Concanavalin A, form aggregates in vitro with rabies virus, whereas limulin had no such effect. Arachis hypogaealectin was only effective in causing agglutination if the virus particles were desialated.
Ziegler and Pozos reported in Infect. Immunity 34: 588-595 (1981) that Concanavalin A binds to the herpes simplex virus and renders it inactive. A similar report with respect to succinyl Concanavalin A was published by Garrity et al. in Antimicrob. Agents Chemotherapy 21: 450-455 (1982).
Olofsson et al in Arch. virol 76: 25-28 (1983) suggested that the affinity of herpes simplex virus glycoprotein for Helix pomatia lectin was due to the presence of N-acetyl galactosamine as terminal sugar in the oligosaccharide of the virion envelope.
Klein's U.S. Pat. No. 4,197,294 disclosed the feeding of vegetable materials, including elderberries and elderflowers of an unspecified species to chickens to increase the iodine, niacin hormones, calcium and magnesium in their diets. There is no disclosure of the presence or use of a lectin.
Sugimoto's U.S. Pat. No. 4,296,025 disclosed the use of a toxic lectin (phytohaemagglutinin) in the laboratory production of interferon.
Yoshii Chemical Abstracts Vol. 49 8402 (e) discusses the reaction of leaf press juices of various plants on a nonenveloped virus (tobacco mosaic virus). Juice from inter alia Sambucus sieboldiana was found to have a powerful inhibitious effect on tobacco mosaic virus infection.
Furassawa in Chemical Abstracts Vol. 74 86207k disclosed the activity of alkaloids derived from bulbs of Narcissus tazetta and from Sambucus sieboldiana against certain viruses. The present invention uses lectins which are glycoproteins, not alkaloids. Alkaloids are generally toxic and not suitable for pharmaceutical use. Those of sambucus plants are found in inedible parts of the plant.
Without wishing to be bound by any theory, we believe that the lectins bind themselves to the virions thereby resulting in agglutination of virus articles which prevent their penetration to cells. It is, however, possible that alternative mechanisms are involved, for example, that the lectins bind to the cell surface thereby blocking virus receptor sites on the cell wall, or that by modifying the surface of the cell wall the lectins act to lock virus into the cell membrane, thereby preventing the release of viral replicates. It is even possible that lectins act in same way to interfere with the intracellular replication of viruses.
The applicant believes it possible that the mode of action of certain traditional herbal or fruit-based remedies from various disorders such as the common cold may have been through lectins.
One purpose of the present invention is to use non-toxic lectins for prevention or treatment of diseases caused by enveloped viruses.
Another purpose of the invention is to provide a pharmaceutical basis and mechanism for the antiviral activity.
Pharmaceutical regimen plant extracts containing some of the lectins to be used in the present invention may have previously found medical use. For example, Sambucus nigra has been used as a diaphoretic, a diuretic and a cathartic agent; Datura stramonium has been used as agent against coughs and laryngitis; Phytolacca has been used as an antirheumatic preparation and as a topical antiparasitic agent. Solanum tuberosum has been used as a spasmolytic agent. We have further identified the composition of the active components of Sambucus nigra 1 agglutinin.